Paper strengthened with solubilized collagen and method

ABSTRACT

A method for making a collagen strengthened cellulosic sheet by providing a cellulosic pulp slurry; adding solubilized collagen to the pulp slurry, and mixing for a time effective for interaction of the cellulosic pulp slurry and solubilized collagen; forming the interacted cellulosic pulp slurry and solubilized collagen into a sheet; and drying the sheet; also, a method for using solubilized collagen for strengthening paper by mixing the solubilized collagen with a cellulosic pulp slurry; and making a cellulosic pulp product from the mixture and drying.

This is a divisional of application Ser. No. 08/250,806 filed on May 27,1994 now abandoned which is a continuation-in-part application of Ser.No. 08/078,932 filed Jun. 16, 1993 now U.S. Pat. No. 5,316,942.

FIELD OF THE INVENTION

This invention relates to a process for making solubilized collagen andfor making solubilized collagen-strengthened paper that providesadvantages over other known processes that make improved papers. Theinvention also relates to the improved solubilized collagen and improvedpaper made by the process. The invention has utility in making low costsolubilized collagen and in binders for cellulosic products, especiallyin the production of recycled cellulosic paper that has improvedmechanical properties and low cost.

The present invention is related to the application entitled RECYCLEPROCESS FOR THE PRODUCTION OF LOW-COST SOLUBLE COLLAGEN having attorneydocket number 21880(1)/2914-1, filed concurrently and having the samefiling date as the present application, the disclosure of which isincorporated by reference herein.

BACKGROUND OF THE INVENTION

The processing of animal hides to produce leather is an ancient art, andtoday it is a very mature industry. Excellent references to thechemistry of leather manufacture by McLauglin, G. D., et al, TheChemistry of Leather Manufacture, Reinhold Publishing Corp, N.Y. (1945),and collagen reactivity by Gustavson, K. H., The Chemistry andReactivity of Collagen, Academic Press Inc., N.Y. (1956), date from the1940's and 1950's, and are still basic descriptions of the art practicedtoday. The name "collagen" is derived from the Greek word for glue, asis the term "colloid" which means "gluelike" in Greek.

Skin is composed of four distinct layers, which are, proceeding fromoutside-in: (1) a thin outer layer of epithelium termed the "epidermis",which is rich in the protein keratin, not collagen; (2) a densecollagen-rich layer, termed the "dermal" or "grain" layer, also calledin the older literature the "thermostat" layer; (3) a thicker layer ofless-dense, collagen-rich connective tissues, termed the "corium" layer;and (4) an inner layer of "subcutaneous tissue", known to the tanner as"flesh", by which the skin is attached to the underlying tissue.

Although hides may merely be "cured" in salt and/or other biocidalsolutions to stop microbial degradation, many hides that are intendedfor use in leather manufacture are "limed", that is, soaked in asaturated solution of hydrated lime (calcium hydroxide) and water. Theliming process initiates the loosening of the epidermis and thesubcutaneous layer, and is the first step in the dehairing process.After liming is complete, the hair, epidermis, and any residual flesh,fat and surface muscles are removed by mechanical scraping, and thedermal layer is mechanically cut, along with enough of the corium layerto give the final leather its required thickness, from the remaininginner corium layer.

In leather-making the primary interest is on the dense collagen-richdermal layer, which is about 25% of the thickness of the corium layer.During the process of leather-making, the dermal tissue receivesseparate chemical and tanning treatments to stabilize the collagenstructure.

The residual portion of the corium layer that is separated from thedermal layer is termed the "limed split" and is a by-product waste ofthe leather manufacturing process. It is these limed splits that become,for example, the collagen-rich feedstock for sausage casing production,and that have been used as the source of collagen for the examplesherein.

During the liming process, the skin imbibes and binds water, and becomeshighly swollen; in the process it acquires a very alkaline pH of about12.5. The chemistry of the liming process is quite well understood.Prior to further leather processing, and in the collagen productionprocess considered here, the skins must be "delimed" by soaking in acidor salt solutions.

Recycling of cellulosic materials to preserve natural resources andreduce costs is presently a desirable environmental objective. Therecycled cellulosic materials are preferably used to replace endproducts where virgin cellulosic materials have historically been used.Unfortunately, products made from recycled cellulosic materials usuallyhave physical characteristics that differ from those made from virginmaterials. One of these important characteristics is strength which isoften significantly reduced.

Previous attempts to provide increased strength to paper include thatdisclosed in the patent to Young, U.S. Pat. No. 3,532,593. Youngdescribes a mechanical method for isolating preexisting gelled collagenfibers, not an enzymatic method for solubilizing the collagen as in thepresent invention. This patent describes a method for removing fat fromcollagen. The collagen is mechanically treated by beating in an acidsolution but remains insoluble. The insoluble mechanically treatedcollagen was then combined with cellulose beaten pulp and made intopaper sheets.

A French journal article by G. Sauret et al, Le collagne ans lafabrication du papier, Revue A.T.P.I., Vol 33, No. 8, Octobre 1979, pp374-365, discloses a mechanical method using a Turmix-Waring blender forpreparing collagen. The mechanically treated collagen is insoluble. Itis combined with cellulose pulp and made into paper sheets.

In contrast, the present invention uses a method that combines smallamounts of soluble collagen with cellulosic material as furtherdescribed herein.

SUMMARY OF THE INVENTION

A typical embodiment of the invention is a method for producing anaqueous solution of solubilized collagen by the steps of (a) providingan aqueous ground slurry of insoluble collagen and adjusting the pH ofthe slurry to obtain activity for a later added proteolytic enzyme; (b)adding the proteolytic enzyme to the pH adjusted slurry; (c) reactingthe slurry and enzyme of step b and\or recycled insoluble collagen andenzyme of step e at a temperature, T, and for a time, t, effective forforming a solution increased in solubilized collagen; (d) addingadditional water and insoluble collagen to the solution of step (c) andmixing; (e) separating at least some of the solution of step dcontaining solubilized collagen from the insoluble collagen, whereby atleast a portion of the insoluble collagen and proteolytic enzyme isrecycled to step c, and a separated solution containing solubilizedcollagen is withdrawn as product. Another typical embodiment does notemploy the recycle step but uses the solubilized collagen directlywithout removal of enzyme. Typically step c may be repeated two, three,four or more times. Additional enzyme may be added to the recycledinsoluble collagen from step e that substantially replaces enzymeremoved with the withdrawal of product or when the rate of reaction onrecycling decreases below a predetermined level. In one typicalembodiment, the method is operated as a continuous process.

The reaction may typically be stopped by adjusting the pH to that wherethe proteolytic enzyme is substantially inactive; and/or by reducing thetemperature to that where the proteolytic enzyme is substantiallyinactive. In another typical embodiment in step a, the liquid or solidscontent of the wet ground slurry is preferably adjusted so that thesolids are at a concentration of about 0.1 to about 1.0 wt %; in step cthe temperature, T, is preferably about 5° C. to about 30° C., and morepreferably about 15° C. to about 28° C. In another preferred embodimentthe solids concentration is between about 0.3 to 0.35 wt % and thereaction of step c is at a temperature of about 10° to about 30° C., andfor a time of 10 to 72 hours; more preferably the temperature is between15° C. and 28° C. Typical proteolytic enzymes are selected from thegroup consisting of porcine mucosal pepsin, bromelain, chymopapain,chymotrypsin, collagenase, ficin, papain, peptidase, proteinase A,proteinase K, trypsin, microbial protease, and combinations of suchenzymes. More preferably the proteolytic enzyme is pepsin or a microbialacid protease. When porcine mucosal pepsin is selected the pH ispreferably about 1.5-3.0, and the temperature about 15° C. to about 28°C. Typically, at least 80 wt % of the insoluble collagen is converted tosoluble collagen with a number average molecular weight 300,000 daltonsand above; while more preferably at least 90 wt % of the insolublecollagen is converted to soluble collagen and the number averagemolecular weight is above 1,000,000 daltons.

A further typical embodiment of the invention includes a method forproducing an aqueous solution of solubilized collagen by the steps of(a) providing an aqueous ground slurry of insoluble collagen; (b)adjusting the water or solid content of the wet ground slurry wherebythe insoluble collagen is at a concentration that promotes substantiallymaximum solubilized collagen concentration in a final product; (c)adjusting the pH of the slurry from step b to obtain activity for aproteolytic enzyme added in step d; (d) adding and mixing theproteolytic enzyme with the pH adjusted slurry; (e) reacting the slurryof step d and\or the recycled insoluble collagen of step g at atemperature, T, and for a time, t, effective for forming a solutioncomprising solubilized collagen derived from the insoluble collagenparticles; (f) adding additional water and insoluble collagen to thesolution containing solubilized collagen in step e and mixing; (g)separating at least some of the solution of step f containingsolubilized collagen from the insoluble collagen and returning theinsoluble collagen to step e, whereby at least a portion of theproteolytic enzyme is recycled, and a separated solution containingsolubilized collagen is withdrawn as product. Another typical embodimentdoes not employ the recycle step but uses the solubilized collagendirectly without removal of enzyme. Typically step e may be repeatedtwo, three, four or more times. Additional enzyme may be added to therecycled insoluble collagen from step e that substantially replacesenzyme removed with the withdrawal of product or when the rate ofreaction on recycling decreases below a predetermined level. In onetypical embodiment, the method is operated as a continuous process. Thereaction may typically be stopped by adjusting the pH to that where theproteolytic enzyme is substantially inactive; and/or by reducing thetemperature to that where the proteolytic enzyme is substantiallyinactive. In another typical embodiment in step b, the liquid or solidscontent of the wet ground slurry is preferably adjusted so that thesolids are at a concentration of about 0.1 to about 1.0 wt %; in step ethe temperature, T, is preferably about 5° C. to about 30° C., and morepreferably about 15° C. to about 28° C. In another preferred embodimentthe solids concentration is between about 0.3 to 0.35 wt % and thereaction of step e is at a temperature of about 10 to about 30° C., andfor a time of 10 to 72 hours; more preferably the temperature is between15° C. and 28° C. Typical proteolytic enzymes are selected from thegroup consisting of porcine mucosal pepsin, bromelain, chymopapain,chymotrypsin, collagenase, ficin, papain, peptidase, proteinase A,proteinase K, trypsin, microbial protease, and combinations of suchenzymes. More preferably the proteolytic enzyme is pepsin or a microbialacid protease. When porcine mucosal pepsin is selected the pH ispreferably about 1.5-3.0, and the temperature about 15° C. to about 28°C.

Typically, at least 80 wt % of the insoluble collagen is converted tosoluble collagen and the number average molecular weight is above300,000 daltons; while more preferably at least 90 wt % of the insolublecollagen is converted to soluble collagen.

Another embodiment of the invention is a method for producing an aqueoussolution of solubilized collagen by the steps of providing an aqueousground slurry of insoluble collagen; adjusting the water or solidcontent of the wet ground slurry whereby the insoluble collagen is at aconcentration that promotes substantially maximum solubilized collagenconcentration that is adapted to strengthen paper in a final product;adjusting the pH of the slurry from Step b to obtain activity for aproteolytic enzyme added in Step d; adding the proteolytic enzyme to thepH adjusted slurry and reacting at a temperature, T, and for a time, t,effective for forming solubilized collagen from the insoluble collagenparticles; controlling the reaction conditions for obtaining a highconcentration of soluble collagen by measuring the concentration ofsolubilized collagen and the molecular weight of the solubilizedcollagen, whereby the reaction is complete when the number averagemolecular weight fraction above 300,000 daltons and the concentrationare substantially maximized; and withdrawing the aqueous solution ofsolubilized collagen as product.

Feed material for the process can typically come from a variety ofsources as long as the feed is relatively clean and has collagencontaining material of relatively small particle size, see for examplethe method of Komanowsky et al discussed below. One typical method forpreparing the feed material of a wet ground slurry of insoluble collagenfrom animal tissues includes the steps:

(a) providing soft animal tissues containing collagen; (b) cleaning thecollagen containing tissues to remove hair, fat, carbohydrates, andother contaminants; (c) cutting the cleaned collagen containing tissuesinto small pieces; (d) mixing the small pieces with water to obtain aslurry; (e) adjusting the pH of the slurry substantially near theisoelectric point of collagen from the tissues; (f) wet grinding theresulting pH adjusted slurry to obtain a slurry of insoluble collagen.The pH of this method is typically about 3 to about 7. The inventionfurther encompasses the unique aqueous solutions of solubilized collagenproduced by the above methods.

A yet further embodiment of the invention includes a method for making acollagen strengthened cellulosic sheet by the steps of:

(a) providing a cellulosic pulp slurry; (b) adding solubilized collagento the pulp slurry, and mixing for a time effective for interaction ofthe cellulosic pulp slurry and solubilized collagen; (c) forming theinteracted cellulosic pulp slurry and solubilized collagen into a sheet;and (d) drying the sheet. Typically the formed sheet may be a sheet suchas paper. Another embodiment includes a method for using solubilizedcollagen for strengthening paper by mixing the solubilized collagen witha cellulosic pulp slurry, molding the mixture and drying.

A still further embodiment includes a strengthened cellulosic pulpcomposition of a dried reaction product of a mixture of solubilizedcollagen and cellulosic pulp. Another typical embodiment is astrengthened paper product of paper prepared from a mixture ofsolubilized collagen and cellulosic pulp.

Yet another typical embodiment includes a method for making a collagenstrengthened cellulosic sheet by the steps of: (a) mixing a cellulosicmaterial selected from the group consisting of virgin paper pulp, broke,reclaimed newsprint, reclaimed carton container, or a mixture thereofwith a solution comprising water, or water and caustic, and mechanicallypulping until a pulp slurry is formed having a consistency of about 3 wt% to about 6 wt % based on dry pulp solids; (b) diluting the pulp slurryto a consistency of about 1 wt % to about 3 wt % based on dry pulpsolids and adjusting pH to about 3.5 to about 7.0; (c) adding betweenabout 0.1 dry wt % to about 2 dry wt % soluble collagen (based on dryweight of cellulosic material) to the diluted pulp slurry, and mixing ata shear rate and a time effective for interaction of the diluted pulpslurry solids and soluble collagen, whereby at least a substantialportion of the soluble collagen is bound to the paper pulp to form acollagen-pulp slurry; (d) diluting the collagen-pulp slurry to betweenabout 0.1 dry wt % and 1 dry wt % consistency; (e) forming thecollagen-pulp slurry into a sheet and drying the sheet. Typically themixing in step c is for about 15 minutes. The pH may be adjusted with anacid selected from the group consisting of muriatic acid, HCl, HNO₃, H₂SO₄, and acetic acid. If desired the method may include the additionalstep of coating the sheet of step e with sizing prior to drying.Typically the sizing further may be a collagen hydrolyzate having anumber average molecular weight of 100,000 daltons or less. The driedsheet may be calendered. Typically the caustic of step a can be a NaOHsolution with a concentration of about 0.25 wt % to about 1.00 wt %based on dry weight of cellulosic pulp solids, and a pH range 10-14.

Typically the solubilized collagen has a number average molecular weightabove 300,000 daltons, and most preferably above about 1,000,000daltons. The mixing shear rate and other conditions are adapted topromote collagen-pulp interactions without denaturation of the collagentriple helical structure. In some applications the collagen-paper slurrypreferably has a consistency of about 0.5 dry wt %. If desired analum/rosin additive is added after pulping in step a or after dilutionin step b or after refining. Also after forming the sheet in Step e, theformed sheet can be wet pressed to a preselected thickness prior todrying.

In one typical embodiment, when only water is selected in step a, theadditional step of refining the pulp/water slurry from Step a ispreferred to fibrillate cellulose fibers in order to obtain a selecteddegree of freeness upon forming a sheet in Step e. When substantiallyreclaimed newsprint is selected, the degree of freeness is preferablybetween about 100 CSF and about 150 CSF and when substantially reclaimedcarton container is selected the degree of freeness is preferablybetween about 300 CSF and about 400 CSF.

A yet further embodiment includes the steps of a method for making acollagen strengthened cellulosic sheet by the steps of:

(a) mixing a cellulosic material selected from the group consisting ofvirgin paper pulp, broke, reclaimed newsprint, reclaimed cartoncontainer, or a mixture thereof with a solution comprising water, orwater and NaOH, and mechanically pulping until a pulp slurry is formedhaving a consistency of about 3 wt % to about 6 wt % based on dry pulpsolids; (b) diluting the pulp slurry to a consistency of about 1 wt % toabout 3 wt % based on dry pulp solids and adjusting pH to about 3.5 toabout 7.0; (c) adding an alum/rosin additive to the pulp slurry afterStep a or to the diluted pulp slurry after Step b; (d) forming thediluted pulp slurry containing alum rosin into a sheet; (e) coating oneor both sides of the sheet with collagen hydrolyzate having a numberaverage molecular weight of 100,000 daltons or less; and drying thesheet.

Another typical embodiment includes a method for making a collagenstrengthened cellulosic sheet by the steps of:

(a) mixing a cellulosic material selected from the group consisting ofvirgin paper pulp, broke, reclaimed newsprint, reclaimed cartoncontainer, or a mixture thereof with a solution comprising water, orwater and NaOH, and mechanically pulping until a pulp slurry is formedhaving a consistency of about 3 wt % to about 6 wt % based on dry pulpsolids; (b) diluting the pulp slurry to a consistency of about 1 wt % toabout 3 wt % based on dry pulp solids and adjusting pH to about 3.5 toabout 7.0; (c) providing an aqueous ground slurry of insoluble collagen;(d) adjusting the water or solid content of the wet ground slurrywhereby the insoluble collagen is at a concentration that promotessubstantially maximum solubilized collagen concentration and molecularweight in a final product; (e) adjusting the pH of the slurry from Stepd to obtain activity for a proteolytic enzyme added in Step f; (f)adding the proteolytic enzyme to the pH adjusted slurry and reacting ata temperature, T, and for a time, t, effective for forming a solution ofhigh molecular weight solubilized collagen from the insoluble collagenparticles; (g) controlling the reaction to obtain a high degree ofsolubilization of collagen and a molecular weight of the solubilizedcollagen where the collagen is capable of binding with cellulosic pulpby simultaneously measuring the concentration of solubilized collagenand the molecular weight of the solubilized collagen, whereby thereaction is complete when the molecular weight and the concentration aresubstantially maximized; (h) adding and insoluble collagen with orwithout additional water to the solution containing high molecularweight solubilized collagen in Step f and mixing; (i) separating atleast some of the solution containing high molecular weight solubilizedcollagen from the insoluble collagen and returning the insolublecollagen to Step d, whereby at least a portion of the proteolytic enzymeis recycled, and the separated solution containing high molecular weightsoluble collagen is withdrawn; (j) adding the separated solution of Stepi. comprising between about 0.1 dry wt % to about 2 dry wt % solublecollagen (based on dry weight of cellulosic material) to the dilutedpulp slurry, and mixing at a shear rate and a time effective forinteraction of the diluted pulp slurry solids and soluble collagen,whereby at least a substantial portion of the soluble collagen is boundto the paper pulp to form a collagen-pulp slurry; (k) diluting thecollagen-pulp slurry to between about 0.1 dry wt % and 1 dry wt %consistency; (1) forming the collagen-pulp slurry into a sheet; anddrying the sheet.

A still further embodiment includes a method for producing a collagenstrengthened sheet by the steps of: (a) providing an aqueous groundslurry of insoluble collagen and adjusting the pH of the slurry toobtain activity for a proteolytic enzyme added in Step b; (b) adding theproteolytic enzyme to the pH adjusted slurry; (c) reacting the slurryand enzyme of Step b or Step e at a temperature, T, and for a time, t,effective for forming a solution increased in high molecular weightsolubilized collagen; (d) adding insoluble collagen with or withoutadditional water to the solution of Step c and mixing; (e) separating atleast some of the solution of Step d containing high molecular weightsolubilized collagen from the insoluble collagen, whereby at least aportion of the proteolytic enzyme is recycled to Step c, and theseparated solution containing high molecular weight solubilized collagenis withdrawn as product; (f) mixing a cellulosic material selected fromthe group consisting of virgin paper pulp, broke, reclaimed newsprint,reclaimed carton container, or a mixture thereof with a solutioncomprising water, or water and caustic, and mechanically pulping until apulp slurry is formed having a consistency of about 3 to about 6 wt %based on dry pulp solids; (g) diluting the pulp slurry to a consistencyof about 1 to about 3 wt % based on dry pulp solids and adjusting pH toabout 3.5 to about 7.0; (h) adding soluble collagen from Step e to thediluted pulp slurry in an amount from between about 0.1 to about 2 drywt % soluble collagen (based on dry weight of cellulosic material), andmixing at a shear rate and a time effective for interaction of thediluted pulp slurry solids and soluble collagen, whereby at least asubstantial portion of the soluble collagen is bound to the paper pulpto form a collagen-pulp slurry; (i) diluting the collagen-pulp slurry tobetween about 0.1 dry wt % and 1 dry wt % consistency; and (j) formingthe collagen-pulp slurry into a sheet and drying.

Another typical embodiment includes a method for producing an aqueoussolution of high molecular weight solubilized collagen by the steps of:(a) providing an aqueous ground slurry of insoluble collagen; (b)adjusting the water or solid content of the wet ground slurry wherebythe insoluble collagen is at a concentration that promotes substantiallymaximum solubilized collagen concentration and molecular weight in afinal product; (c) adjusting the pH of the slurry from Step b to obtainactivity for a proteolytic enzyme added in Step d; (d) adding and mixingthe proteolytic enzyme with the pH adjusted slurry; (e) reacting theslurry of Step d at a temperature, T, and for a time, t, effective forforming a solution comprising high molecular weight solubilized collagenderived from the insoluble collagen particles; (f) adding additionalwater and insoluble collagen to the solution containing high molecularweight solubilized collagen in Step e and mixing; (g) separating atleast some of the solution of Step f containing high molecular weightsolubilized collagen from the insoluble collagen and returning theinsoluble collagen to Step e, whereby at least a portion of theproteolytic enzyme is recycled, and the separated solution containinghigh molecular weight solubilized collagen is withdrawn as product; (h)mixing a cellulosic material selected from the group consisting ofvirgin paper pulp, broke, reclaimed newsprint, reclaimed cartoncontainer, or a mixture thereof with a solution comprising water, orwater and NaOH, and mechanically pulping until a pulp slurry is formedhaving a consistency of about 3 to about 6 wt % based on dry pulpsolids; (i) diluting the pulp slurry to a consistency of about 1 toabout 3 wt % based on dry pulp solids and adjusting pH to about 3.5 toabout 7.0; (j) adding soluble collagen from Step e to the diluted pulpslurry in an amount from between about 0.1 to about 2 dry wt % solublecollagen (based on dry weight of cellulosic material), and mixing at ashear rate and a time effective for interaction of the diluted pulpslurry solids and soluble collagen, whereby at least a substantialportion of the soluble collagen is bound to the paper pulp to form acollagen-pulp slurry; (k) diluting the collagen-pulp slurry to betweenabout 0.1 dry wt % and 1 dry wt % consistency; and (l) forming thecollagen-pulp slurry into a sheet and drying.

A further embodiment of the invention includes a method for making acollagen strengthened cellulosic sheet by the steps of:

(a) providing a cellulosic pulp slurry; (b) adding solubilized collagento said pulp slurry whereby said cellulosic pulp and said solubilizedcollagen have a consistency above about 2 wt %, and mixing for a timeeffective for interaction of said cellulosic pulp slurry and solubilizedcollagen and whereby said mixing is at a temperature above about 35° C.,or more preferably above 40° C.; (d) forming said interacted cellulosicpulp slurry and solubilized collagen into a sheet; and (e) drying saidsheet.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a plot showing the non-Newtonian behavior of the collagensolutions. Viscosity of diluted solutions of solubilized collagen of theinvention (A) and BA-1 collagen solutions (B) at two shear rates (20 and100 rpm). Viscosity, in centipoise, is plotted in the ordinate (verticalscale) and the approximate solids concentration, in mg/mL, is plotted inthe abscissa (horizontal scale).

FIG. 1B is a plot showing the ratio of the viscosity determined at 20rpm to the viscosity at 100 rpm, termed here the "viscosity ratio". Thedata is calculated from the data in FIG. 1A for both solubilizedcollagen of the invention (A) or the BA-1 collagen solutions (B). Theviscosity ratio is plotted in the ordinate (vertical scale) and theapproximate solids concentration, in mg/mL, is plotted in the abscissa(horizontal scale).

FIG. 2 is a plot of the data for Example 1A showing the viscosity at 20rpm and 100 rpm. Viscosity, in centipoise, is plotted in the ordinate(vertical scale) and the duration of the reaction, in hours, is plottedin the abscissa (horizontal scale).

FIG. 3 is a plot of a small-scale batch collagen solubilizing reactiondemonstrating the pepsin recycle of Example 3A. The viscosity ratio isplotted in the ordinate (vertical scale) and the time of reaction, inhours, is plotted in the abscissa (horizontal scale).

FIG. 4 is a plot of the development of viscosity ratios in Examples 5A(denoted by A) and 6A (denoted by B). The viscosity ratio is plotted inthe ordinate (vertical scale) and the time of reaction, in hours, isplotted in the abscissa (horizontal scale).

DETAILED DESCRIPTION OF THE INVENTION

It was recognized that solubilized collagen material, added to acellulose pulp prior to the papermaking process (i.e, mixed with thecellulose pulp fibers in the machine chest), resulted in a significantincrease in strength of the paper-collagen composite. This result issurprising since the prior art teaches that larger insoluble aggregatesof collagen, such as those produced by mechanical diminution of bovinehides, are necessary. One reason that the use of soluble collagen inpapermaking may not have been considered is that soluble collagen can beexpected to thermally denature at the fluids temperatures employed inpapermaking (greater than about 40° C.). Denatured collagen is notexpected to be as useful as native collagen aggregates. It is furthersurprising since one would expect that the cellulose pulp could best bebound together by larger size particles such as those of the scale ofthe cellulose pulp itself and not those that are soluble in water. As isdemonstrated in the examples herein solubilized collagen that had beencentrifuged at very high gravitational forces that would removesubstantially all insoluble materials was very effective in increasingthe strength of paper. Further, there is no current large-scale use orcommercial source for a cost effective collagen solution of this type.Small-scale applications for soluble collagen exist in the food,cosmetic and pharmaceutical industries, for which the products are muchhigher priced than will be economically acceptable in the cellulose pulpand paper applications of the invention.

DEFINITIONS

The following definitions will be useful in reading the disclosureherein:

Acidified collagen--collagen that has been treated with an acid orextracted by an acid solution.

Beating--mixing paper pulp at a relatively high shear rate in order toseparate and expand the size of pulp fibers.

Broke--scrap paper from the papermaking process.

Calendering--process of creating surface smoothness and hardness inpaper typically by on-line compression between (counter-rotating)cylinders.

Cellulosic pulp--fibers from cellulosic materials that could be wet ordry and produced by mechanical, chemical or other means.

Collagen gel--collagen that exists in its native molecular state in acontinuous, highly hydrated fibrillar network.

Collagen sizing--collagen added as a coating after paper sheet has beenmade.

Degree of freeness--a measure of how easily (freely) water will drainfrom a paper sheet during production, performed in a standardized testapparatus; one industry recognized standard is Canadian StandardFreeness (CSF).

Mechanically pulping--mechanical separation of cellulosic fibers byspecially designed high-shear mixers.

Mechanically working--mechanical shearing of collagen-rich materials toreduce particle size and initiate gel formation.

Mixing collagen and cellulosic (e.g. paper pulp)--mixing is at arelatively lower shear rate (as compared to beating) that is conduciveto the reaction of higher molecular weight collagen with cellulosic pulpso as to obtain interaction of solubilized collagen and cellulosic pulp.

Molecular weight--this term as used herein is intended to refer tonumber average molecular weight unless otherwise specified.

Natural or native collagen--collagen molecules that retain the normaltriple-helical assembly of alpha-chains.

Old corrugated container--secondary cellulosic fiber from recycledcorrugated container or similar Kraft pulping process sources.

Old newsprint--secondary fiber from recycled newspapers and similarsources.

Reclaimed paper--paper as received from recycling operations.

Recycled paper--reclaimed paper that has been reprocessed and made intonew usable paper.

Refining--a pretreatment for the paper pulp that expands and separatescellulosic pulp fibers.

Solubilized collagen--collagen that has been treated to separate thecollagen fibrils to render them soluble while retaining the normaltriple-helicol assembly of native collagen; covalent bonds betweencollagen fibrils are broken so that smaller collagen molecules can gointo solution; this is in comparison to mechanically worked and or acidtreated collagen that merely makes the collagen pieces physicallysmaller but does not break the covalent bonds between fibrils; thesolubilized collagen used herein has been solubilized by an enzymatictreatment that breaks the covalent bonds between collagen fibrils.

Viscosity ratio--the ratio of two viscosity measurements of a solutionat two different shear rates. This is one typical way to follow theincrease or decrease of viscosity due to an increase or decrease ofsolubilized collagen being produced from a slurry of insoluble collagen.Another typical method would be to use only the viscosity measurement tofollow the increase or decrease of solubilized collagen.

A. FIRST GENERAL EMBODIMENT

One typical embodiment of the first general embodiment achieves lowercosts of operation by utilizing recycle steps to recapture and reuseenzyme that would normally be lost on removal of soluble collagenproduct solutions. Another typical embodiment of the first generalembodiment also has low costs of operation but does not utilize therecycle steps to recapture enzyme. In this latter embodiment thesolubilized collagen is sent directly to its end use, such as inpapermaking, with no attempt to remove enzyme or otherwise purify thesolubilized collagen.

Advantages of the first general embodiment of the invention are in: (1)minimizing the cost of preparing soluble collagen by processing directlyfrom ground skin material to the maximum amount of solublemacromolecules; and (2) at the same time, maximizing the degree ofconversion to soluble collagen material capable of binding to cellulosicpulp and controlling the molecular weight of the soluble collagenmaterial in order to enhance the binding effect to the pulp fibers,thereby maximizing the resulting tensile strength and/or othermechanical properties of the paper product. Another major advantage ofusing solubilized collagen over the insoluble larger aggregates of theprior art, in the production of cellulosic products such as paper, isgreater uniformity in the distribution of collagen in the cellulosicpulp.

Bovine skin was selected as the collagen source in the examplesdescribed here because collagen preparation methods from skin have beenwidely reported, and the material is a high volume by-product of themajor industries of beef production and leather manufacture; however, itis expected that collagen obtained from other sources (e.g. tendon) willwork in the process also.

Collagen solubilization of skin has been accomplished by an enzymatichydrolysis process with an animal stomach enzyme (e.g. pepsin) andseveral other enzymes without any other purification steps. The processresults in nearly complete solubilization of ground hide preparations in10-30 hours at room temperature in acidic solutions. Other (untested)enzymes may yield faster or cheaper conversion of collagen-containingtissues, and the process has not necessarily been optimized to minimizeenzyme requirements and production time. To date, the process has beenscaled to produce approximately 500 gallons of 0.3-0.4% collagensolution, and it has been demonstrated to be relatively easy to control.

EXAMPLES

The following examples, illustrative of the novel compositions and thenovel methods of preparing them, are given without any intention thatthe invention be limited thereto.

Materials

The pepsin used was a crude (relatively unpurified) powder from pigstomach mucosa (Cat. No. P7125) purchased from Sigma Chemical Company,St. Louis, Mo. Lot # 070H0437 of this product, used in the examples,contains approximately 15% protein (by UV), with an activity of 91pepsin units/mg solids and 620 units/mg protein. Residual solids in thepreparation appear to be a combination of precipitation salts, buffersalts and/or carbohydrates. Crystallized pepsin has a maximum specificactivity of about 3500 units/mg protein.

Additional tests were performed with pepsin, crude powder, from SigmaChemical Company; AFP 2000, acid fungal protease from a strain ofAspergillus niger, from Solvay Enzymes; Newlase A from a strain ofAspergillus niger, and Newlase II (from a strain of Rhizopus niveus,from Amano Enzyme U.S.A.; Quest AP, quest acid protease from a strain ofAspergillus niger, from Quest International; EDC-APA, an EDC acidprotease A, and EDC-APB, an EDC acid protease B, from Enzyme DevelopmentCorporation.

The collagen slurry used herein for Examples 1A-6A was prepared fromground limed-splits of bovine skin. The collagen was supplied byTeepak's Sandy Run Plant, Columbia, S.C. Typical analyses for thematerial of Example 6A are pH=6.4; solids content=15.67%; gelatincontent=2.62%; fat content=2.1%. A 1974 USDA report by Komanowsky, M.,et al, "Production of Comminuted Collagen for Novel Applications", J.American Leather Chem. Assoc., 6, 410-422 (1974), describes techniquesfor pre-slicing, acidifying and wet-grinding of limed splits to producefive "comminuted" (ground) collagen products, classified by extent ofgrinding and the resulting particle size and texture. A subsequent 1978paper by Turkot, et al , "Comminuted Collagen: Estimated Costs ofCommercial Production", Food Tech., 48-57 (April, 1978), presents aneconomic analysis of the production costs for these same five products.The output from this plant closely approximates the ground limed-splitmaterial used as a source for collagen in the examples herein.

For Examples 7A to 11A unless otherwise provided the enzymatic collagensolubilization was performed as follows. The collagen source (eitherground whole hides or ground limed splits) ground as described inExample 7A with an 0.06 inch cutting head and in a water slurry was spunat 4° C. in a Beckman J2-21 Centrifuge (JA-20 rotor) at 10,000 rpm for20 minutes to remove excess liquid. This centrifuge provided a ratio ofrpm to gravitation force of about 1:1, thus at 10,000 rpm the G forceswere about 10,000× gravity. The supernatant liquid was removed and thecentrifuged solids (7.5 g) were added to a one liter Erlenmeyer flaskthat contained deionized water (750 mL). The suspension was stirred witha two inch magnetic stir bar and the pH was adjusted using concentratedhydrochloric acid. The enzyme was then added to the flask, which wasplaced in an incubator set to the desired temperature. Viscositymeasurements were made by pouring approximately 100 mL of each reactionmixture into a beaker and bringing to room temperature. The viscositywas measured with a Brookfield Synchro-lectric Viscometer model RVT.Measurements were made at 20 rpm and 100 rpm with spindle No. 3. Threereadings were taken at each speed and averaged for the calculation ofviscosity in centipoise. Aliquots were removed for viscositymeasurements at specified times and then returned to their originalflasks.

A collagen solution ("BA-1"), used as a control solution in theexamples, was supplied as the soluble skin product, Secolan BA-1, byKensey Nash Biomaterials, Exton, Pa. The collagen solution is typicallya white milky color; pH=3.1-3.3; total solids=1% ±0.2%; activecollagen>0.67% (nominally 1% in the examples). This product is sometimesfound to be slightly gelled upon receipt. However, based on the patternobserved after electrophoretic analysis, it is believed that the BA-1 isproduced by an acid-extraction process, not by an enzymatic reaction aspracticed in the present invention.

It was found that the solubilization of collagen-containing solids canbe effectively monitored by periodic measurement of the solutionviscosity . Fluid viscosities can be conveniently measured by a varietyof relatively simple methods, such as the Brookfield Model #RVTViscometer (#3 Spindle) used with the examples. In this Brookfieldsystem, the force exerted by a fluid upon a disk, which is rotated atconstant rotational speed in the fluid, is used to estimate the fluidviscosity. In the collagen solutions described herein, the fluidviscosity will be strongly dependent on the concentration of dissolvedcollagen, the molecular weight distribution of the soluble collagen andthe fluid temperature, and, to a lesser extent, fluid pH and ionicstrength.

When the viscosity is independent of the applied force (shear), then thefluid is said to be "Newtonian". For solutions of many macromolecules,including the rod-like collagen molecules considered here, the solutionviscosity is very dependent on the force applied to the liquid, and theliquid is said to be "non-Newtonian". When the dissolved macromoleculesare highly elongated, and the shear rate (proportional to the rotationalspeed) is sufficiently high, the molecules tend to orient with thestreamlines of the fluid and their effect on the fluid velocity tends todecrease in a manner that is strongly dependent on the shear rate.

The non-Newtonian behavior of collagen solutions is demonstrated in theexperiments summarized in FIG. 1A, in which the viscosity ofpreparations of solubilized collagen and BA-1 were determined at roomtemperature as the solutions were progressively diluted with distilledwater. Some uncorrected increase in solution pH may have occurred inthis experiment as the samples were diluted; however, the trend for thedata is valid.

For each solution, the viscosity was determined at two rotationalspeeds, 20 and 100 rpm. The open circles () and filled circles ()represent data for solubilized collagen of the invention at 20 rpm andat 100 rpm, respectively. The open squares () and the filled squares ()represent the data for the BA-1 collagen control at 20 rpm and 100 rpmrespectively. Both solutions were more viscous at the lower rotationalrate, as expected. The viscosities of the collagen produced in theexamples and BA-1 preparations were substantially different, with theproduced collagen solution having a much higher viscosity at lowercollagen concentrations and a steeper slope. These effects appear to beprimarily due to the difference in the average molecular weights of thecollagen molecules in the two solutions, with the collagen solution ofthe invention having the larger number average molecular size. Thecomparison shows that the method of the invention was successful inmaking a higher viscosity collagen material at a lower concentrationthus showing the number average molecular weight was higher.

The ratio of the viscosity determined at 20 rpm to the viscosity at 100rpm, termed here the "viscosity ratio", is a convenient measure of thisnon-Newtonian, molecular-weight-dependent effect. This is illustrated inFIG. 1B, in which the viscosity ratio is higher for collagen solutionsof the invention than for BA-1. In FIG. 1B the open circles () representdata from the solubilized collagen of the invention and the open squares() represent data from the BA-1 collagen solution. The viscosity ratioused herein is a measure of the "degree of conversion" of solid collagenmaterials to soluble collagen molecules, and also a measure of molecularweight, where higher values of the viscosity ratio will correlate withthe desired higher number average molecular weights of the dissolvedcollagen. In FIG. 1B it is important to note that since the material isbeing diluted, an increase in viscosity ratio is measuring the increasein concentration of soluble collagen since the molecular weight of thematerial remains the same. In tests of the examples below, changes inthe viscosity and viscosity ratio will be measuring changes inconcentration. If desired the peak soluble collagen content can bemeasured by chromatographic and electrophoretic techniques.

Alternatively, analysis of solubilized collagen composition wasroutinely performed by SDS polyacrylamide gel electrophoresis (PAGE)that used a 3% stacking gel; 6% running gel, following denaturation byboiling with β-mercaptoethanol. Some irreversible precipitation occursduring the denaturation process. Gels were stained by Coomassie Blue dyeand destained in staining buffer only.

PAGE results from this technique demonstrate (results not shown here)that BA-1 solutions contain predominately tropocollagen monomer (300,000daltons) aggregates. Collagen solutions produced by the present processthat had acceptable paper binding properties appeared to have a numberaverage molecular weight of at least 300,000 daltons, with somecomponents having the intact triple helix of alpha, beta and gammachains as evidenced by PAGE, other preparations may have had a disruptedhelix.

Analysis of solubilized collagen composition was also routinelyperformed by SDS polyacrylamide gel electrophoresis (PAGE) with thePharmacia PhastGel System. PhastGel Gradient 4-15% polyacrylamide gelswere used. The buffer system in the gel is 0.112M Tris acetate, pH 6.4.PhastGel SDS Buffer Strips that contain, at pH 8.1, 0.2M Tricine, 0.2MTris, and 0.55% SDS were used to run the gels. The separation method wasfrom the PhastSystem Separation Technique File No. 130, Table 2.

Samples were prepared for Gel Electrophoresis by the additionconcentrated stock solutions of SDS (20%) and buffers (5×stock). Thefinal concentrations were 10 mM Tris/HCl (pH 8.0), 1 mM EDTA, 2-2.5%SDS, and 0.01% bromophenol blue. Each sample was then heated at 100° C.for 5 minutes and approximately 1 μL was applied to the gel. In someearly experiments, 2-mercaptoethanol (a reducing agent) was added to thesample before heating. The addition of the 2-mercaptoethanol had noeffect on the gel pattern.

At the completion of the electrophoresis, the gel(s) were stained withthe Pharmacia Silver Kit. The staining method used was from thePhastSystem Silver Kit Instruction Manual , Table 2. The Developmenttime and Background Reduction time were doubled for better visibility onthe gels.

The SDS detergent in the gels disperses all non-covalent collagenaggregates leaving only covalently joined molecules. The degree to whichthese molecules migrate on a gel is related to their molecular weightsand approximate molecular weights have been assigned to the collagenbands by co-electrophoresis of molecular weight standards on the samegels. PAGE analysis of solubilized collagen indicates bands at ˜100,000daltons (alpha-collagen), ˜200,000 daltons (beta-collagen), ˜300,000daltons (gama-collagen), and bands>300,000 daltons. The intensity of thebands is in inverse order of their molecular weights.

Analysis for soluble or insoluble collagen was typically performed byfirst measuring the amount of hydroxyproline in the sample, thencorrelating this concentration with the collagen. Hydroxyproline wasmeasured on 0.1 mL samples that were dried in polypropylene tubes at125° C. The samples were dissolved in 0.05 mL 4M sodium hydroxide,capped, and then autoclaved for 30 minutes. Citric acid (0.05 mL of a1.4M solution) and chloramine T reagent (1 mL of a solution thatcontained 1.41 g chloramine T, 10 mL 1-propanol, 10 mL deionized water,and 30 mL of a pH 6 citric acid/acetic acid buffer) were added to eachtube which was then incubated for 20 min. at room temperature. PDABsolution (1 mL of a solution that contained 15 gp-dimethylaminobenzaldehyde, 62 mL isopropyl alcohol, and 26 mL 60%perchloric acid) was then added. The samples were incubated at 65° C.for 20 minutes, after which time 0.2 mL of each sample was transferredto a micro-titer plate reader and the absorbance read at 570 um. Asample of purified collagen (Vitrogen 100™; Celtrix) that contained 3.0mg/mL collagen was found to contain 0.33 mg/mL hydroxyproline. Usingthis collagen preparation as a standard, multiplication of thehydroxyproline concentration by a factor of 9.1 will yield the collagenconcentration.

High pressure liquid chromatography (HPLC) was performed to analyze theintact soluble collagen molecular weight distribution. HPLC wasperformed with a TOSOHAAS TSK-GEK G6000PW column (30 cm×7.8 mm) on aWaters 650 Advanced Protein Purification System. Absorbance wasmonitored at 220 mm with a flow rate of 0.25 mL/Min. (unless notedotherwise). The mobile phase contained 10 mM hydrochloric acid. A columnprefilter was used with a 10 um frit.

Eluent fractions containing the HPLC peaks were analyzed by PAGEelectrophoresis to determine the size of the constituent collagenmolecules. The SDS in the gels disrupts the collagen aggregates so thatonly the molecular weights of covalently attached molecules can bedetermined by this method. The first eluting peak (Peak 1) containsmolecules with number average molecular weights greater than 300,000daltons as well as molecules with number average molecular weights ofapproximately 200,000 daltons and approximately 100,000 daltons. Thesmaller molecules appear to be constituents of larger aggregates thatwere disrupted by the SDS. The second eluting peak (Peak 2) containedmolecules with number average molecular weights of approximately 300,000daltons, approximately 200,000 daltons and approximately 100,000daltons. The 200,000 dalton and 100,000 dalton molecules appear to bepart of 300,000 aggregates that were disrupted by the SDS detergent. Thethird eluting HPLC peak (Peak 3) contains collagen fragments with numberaverage molecular weights less than approximately 100,000.

In the examples below, it was determined that ground limed splits ofbeef hide can be nearly completely solubilized when they are subjectedto pepsin hydrolysis at pH in the range of 2.0-2.2. Batch reaction timesare typically 10-30 hours at room temperature (22°-26° C.). The maximumconcentration of soluble collagen typically produced in this process isapproximately 0.30-0.40% (3-4 mg dissolved collagen/ml). The process hasbeen demonstrated at up to 2.0 liter-scale and, using essentially thesame recipe, at approximately 500-gal scale, as discussed below.Microbial proteases gave similar results as discussed below.

EXAMPLE 1A

Approximately 15 g of wet Teepak collagen solids were suspended bymagnetic stirrer in 750 ml of Columbus, OH tap water at roomtemperature. The solution pH was adjusted to 2.1 with concentratedhydrochloric acid (HCl)--approximately 65-70 drops. Crude pepsin powder(0.38 g) was then added with stirring into the collagen suspension toinitiate the reaction. The suspension was stirred overnight, duringwhich heating of the solution to 26°-27° C. or higher sometimes occurreddue to conduction from the stirrer plate. The viscosity of the solutionwas measured (20 & 100 rpm) periodically during the second day of thereaction until a maximum in the viscosity ratio was achieved, at whichtime the solution was stabilized by increasing the pH to 3.0-3.5 and/orby placing the solution in the refrigerator. Increasing the pH above 4.0may initiate irreversible gelation of the collagen solution.

Results for Example 1A are plotted in FIG. 2. FIG. 2 shows a plot ofviscosity, (in centipoise) as a function of time reaction (in hours).Viscosity measurements were taken at 20 rpm (squares) and 100 rpm(circles). After completion of the reaction at pH 2.1, three sampleswere taken and the pH adjusted to 2.1 (), 2.8 (), and 3.5 (). Viscositytests at 20 rpm taken several days later confirmed that the samples atpH=3.5 were indeed more stable and retained more of the originalviscosity than those at pH=2.1.

EXAMPLE 2A

Hydrolysis of Teepak collagen at temperatures between 30°-35° C. wasinvestigated in a series of approximately 10 experiments to determinethe potential for minimizing pepsin usage in the solubilization process.Typically, enzyme-catalyzed reaction rates will double with every 5°-10°C. increase in temperature. In these experiments, a 4-liter stainlesssteel beaker was wrapped with heating tape, then insulated with asbestostape. The solution temperature was controlled by a Variac in line withthe heating tape to about ±1°-2° C. The process above was scaled to 2liters of reaction volume, and a range of lower pepsin concentrationsand heating profiles was investigated. In nearly all cases, completesolubilization of the Teepak solids was accomplished in 10-15 hours, andin no case was substantial viscosity developed in the solubilizedproduct.

Typical of the ten experiments is the following: 2 liters of water wereadded to a beaker, to which was added 40 g of Teepak collagen, then thepH was adjusted to 2.13 with concentrated HCl, and finally 1.0 g crudepepsin was added. Initially the bath temperature was 30.0° C., about 2.5hours later the temperature was 33° C. and the viscosity at 100 rpm was19 cps, and about 5.5 hours later the temperature was 36.5° C. with aviscosity of 8 cps. The sample was completely solubilized in less than 8hours at 33°-36° C. with no increase in viscosity indicating theproduction of a higher molecular weight material. These experimentsdemonstrate that it is expected to be more difficult to conserve pepsinin this process by operating at higher reaction temperatures, even earlyduring the hydrolysis process. The maximum feasible temperature foraccumulating this particular large molecular weight collagen appears tobe about 30° C.

EXAMPLE 3A

Another approach for minimizing pepsin usage in the process isillustrated by the experiment summarized in FIG. 3. In this experiment,the recipe above (750 ml Columbus, Ohio tap water, 15.5 g teepakcollagen, 0.38 g pepsin, pH=2.1) was mixed on Day 0 to initiate thereaction in a 2-liter flask at room temperature (Roman numeral I). Afterapproximately 1 day, an additional 750 ml of water and another charge ofTeepak collagen solids (16.1 g) were added, but no additional pepsin wasadded to the reactor (Roman numeral II). The flask was stirred for about5 minutes to mix the contents and the pH was readjusted with 30 drops ofconcentrated HCl, then the stirrer was turned off and the solids werepermitted to settle out. After approximately 30 minutes, 750 ml ofsupernatant, "Day 1" supernatant (D1), was decanted into another flask,and stirring of both flasks was resumed. The Day 1 Supernatant containedsome fine collagen particles, but it contained a much lower suspendedsolids load than the bottom fraction. The same process of dilution (755ml water), collagen solids addition (15.2 g Teepak collagen), pHadjustment with 30 drops concentrated HCl (Roman numeral III), andsupernatant decanting of "Day 2" supernatant (D2) was repeated in thefirst flask after approximately 2 days of reaction.

The progression of the hydrolysis reaction is illustrated by the solidlines (-x-) in FIG. 3. The circles () show a plot of the progressionhydrolysis reaction of the Day 1 supernatant while the squares () show aplot of the Day 2 supernatant. In this example three typical charges ofTeepak collagen were hydrolyzed by a single charge of pepsin, althoughthe rate of hydrolysis appears to be decreasing with each cycle. Becausethe viscosity ratios of both the Day 1 and Day 2 supernatants appearedto increase after they were decanted from the main reactor, it wasapparent that some pepsin and insoluble collagen was transported alongwith the supernatant. However, it appears that the pepsin has a higheraffinity for solid collagen particles than for soluble collagen, thusmost of the enzyme can be recycled several times before it is removedfrom the system, thereby minimizing the cost of this reagent. Preferablybetter separation of liquid and solids is obtained if the supernatant isseparated from the insoluble collagen by centrifugation.

Most preferably a steady state in the processing recycle steps isdesired. This is achieved by adding additional enzyme after the productremoval step, when the rate of reaction in the recycle steps decreasesbelow a predetermined level. Most preferably, additional enzyme is addedthat just replaces that lost with the removal of product.

EXAMPLE 4A

An experiment was conducted in which 750 ml whitewater (recycle waterfrom a papermaking process) was substituted for the tap water in thestandard recipe of Example 3A above. Then 15.5 g Teepak collagen wereadded, the pH was adjusted to 2.14 with 40 drops of concentrated HCl,and 0.375 g of pepsin were added. Because the room temperature waselevated during this experiment, the reaction was conducted at 29°-31°C., and the solubilization appeared to proceed more quickly thanstandard reactions at 25°-26° C. In this single reaction, good viscositywas developed, the solids were nearly completely solubilized, and thereappeared to be no problem with conducting the process in this solution(see Table 1A). Recycling whitewater from a papermaking process in thisway will greatly diminish the amount of water introduced to the process.

                  TABLE 1A                                                        ______________________________________                                        Solubilized Collagen Made                                                     in Whitewater From Paper Making                                               Time     Viscosity        Viscosity                                           (Hours)  20 rpm       100 rpm Ratio                                           ______________________________________                                         0       --           --      --                                              18.5     415          177     2.34                                            22       440          186     2.37                                            26.7     365          166     2.20                                            42       280          136     2.06                                            ______________________________________                                    

EXAMPLE 5A

In this example, 500 gal of Savannah, Ga tap water was delivered to adouble-paddle, 600 gal. stainless steel tank, and 75# of Teepak collagen(13.5# solids @ 18% solids) was dispersed in the water. Approximately1.4 liters of concentrated HCl was added to bring the pH to 2.14. Pepsin(1.01 kg; Sigma Lot # 70H0437) was slowly added, then the tank wascovered with polyethylene film and the tank was stirred overnight. Afterapproximately 20 hours, hydrolysis was incomplete (viscosityratio=1.32). Because the liquid and room temperatures were relativelylow (approximately 20° C.), it was decided to attempt to raise theliquid temperature by putting live steam onto the outside bottom of thetank. The steam was used for about 2.5 hours, by which time the liquidtemperature was 23° C., the viscosity ratio was 2.15, and the steamheating was discontinued.

At approximately 31 hours, the viscosity ratio was 2.43, which isrelatively high for this reaction. It was decided to adjust the pH inthe tank to approximately 3.0, by the addition of approximately 450grams of NaOH flakes, in order to stabilize the solution (slow/stop thepepsin reaction) for use in paper the next day. Approximately 55 gal ofthe pH=2.1 solution were saved in 5-gal containers prior to the pHadjustment. Because the viscosity ratio dropped slightly overnight forthe pH=2.1 solution (open circles, , in FIG. 4 and denoted by A)compared to the pH=3.0 solution (closed circles, ), it is concluded thatpH adjustment is helpful in maintaining the highest possible molecularweight in the product during storage at room temperature.

After approximately 24 hours of reaction, some floating solid material(presumed to be fat because of its low density) was observed on theupper surface of the collagen solution near the mixer shaft. Although noattempt was made in this experiment to remove this residue, it can beeasily skimmed from the preparation if the residual fat was found to bedetrimental to collagen performance.

Prior to using the collagen solution made in this example and in Example6A, described below, the solution was filtered by passing it through aknitted plastic screen with openings approximately 1×3 mm, in order toremove a small number of very slowly degrading skin particles. Theseparticles are characteristically the last material to be dissolved bypepsin and can often be found in the 3-5 mm size range. A large sampleof these residual particles was filtered from the collagen solution andtheir dry weight was measured. Based on projecting this sample to theentire batch of collagen solution, it was estimated that more than 95%of the initial solids were solubilized in this process.

EXAMPLE 6A

In this example, the same tank was filled with 500 gal of Savannah, Ga.tap water, which in January was very cold--about 11° C. Teepak collagen(79.5#; 12.5 # of solids at 15.67% dry wt.) was dispersed in this water,then 1.5 liters of concentrated HCl was added to bring the pH to 2.18.Pepsin (1.01 kg; Sigma Lot # 70H0437) was slowly added, then the tankwas covered with polyethylene film. Live steam was placed on the outerbottom of the tank for approximately 4 hours to raise the liquidtemperature from 11.5 to 25° C. At this time the pH was 2.40; anadditional 0.4 liters of concentrated HCl was added to bring the pH downto 2.29. The tank was draped with polyethylene film to insulate the tankovernight. After approximately 28 hours the viscosity ratio was 2.51,with the temperature at about 22° C. at pH=2.46. Approximately 600 g offlaked NaOH was added to bring the tank contents to pH=2.98, the tankwas covered as before and stirred overnight. The final viscosity ratiowas 2.61. Results are shown in FIG. 4 at B (-x-).

Since the collagen solution in Example 6A was produced at about a 2°-3°C. higher reaction temperature during the first day than that in Example5A, the reaction appears to have progressed more rapidly, reachingcompletion about 4-5 hours sooner. When the pH was adjusted to about 3.0the final solution appears to have slowed the enzymatic reaction so thatlittle degradation of the soluble product was observed overnight.

The process is intended to produce nearly complete conversion of beefhides to a collagen solution using an enzymatic hydrolysis reaction.Objectives for the process are production of soluble collagen product atthe maximum yield, while conversion costs and fixed capital expendituresare minimized. The process is not intended to produce food ormedical-grade soluble collagen, and therefore requirements forproduction of clean solutions are minimal, and no purification of thesoluble collagen is anticipated. No attempt has been made to remove theremnants of the other skin components (fat, proteoglycans, otherproteins, salts, etc.), which are present in the ground-split feedstockat concentrations lower than collagen.

The process will require a series of cutters and grinders to reduce thefeedstock limed splits to a shredded material that can be readilyconverted to soluble collagen. As cited above, the "front end" of theprocess will likely look similar to the USDA process for producingcomminuted collagen. Depending on the pretreatment of the hides employedto prevent microbial growth, the hides may need to be delimed oracidified to remove residual calcium salts or other biocides. The groundsolids are then mixed with process water (perhaps a reduced-solidswhitewater stream from a paper plant), the pH is titrated to 2.0-2.2,and enzyme is added to begin the solubilization process. Followingconversion, the soluble solids can be pumped directly to a paper makingprocess and mixed with refined pulp solids or stabilized and stored.

In small-scale tests, maximum interaction between collagen and pulpsolids appears to result if the pH of the solution is about 4.0 or lessand the pulp consistency is 1.0% or lower. Therefore, adjustment of thepulp in the holding tank to about pH 4.0 or less appears to bebeneficial although a typical run was at pH 5-6 because the paper wasmore stable.

EXAMPLE 7A

"USDA" feed collagen materials were prepared using the method ofKomanowsky et al., cited herein, as follows. Two limed splits and onedehaired and limed hide were rolled up and cut to yield 12 inch widestrips. These strips were passed through a strip cutter and then througha rotary knife cutter, An acidic solution was prepared by dissolving102.15 g of benzoic acid in 1021.5 g of propionic acid. Acidificationwas carried out in 55 gallon stainless steel tumbling drums by adding203 lbs of water and 521 g of the above acid solution to the materialfrom the limed hide splits and 235 lb of water and 603 g of acidsolution to the whole hide material. The drums were tumbled 15 minutesper hour for four hours. The final pH values were 5.1 and 5.2,respectively. Finally, part of both materials was passed through a 0.06inch cutting head of the Urschel Comitrol. The remaining part was passedthrough an 0.200 inch cutting head. The products were poured into smallplastic bags and placed into a freezer at -20° C. for later use.

EXAMPLE 8A

USDA ground limed splits were centrifuged at 4° C. for 20 minutes at10,000 rpm. The supernatant liquid was removed and the centrifuged limedsplits (15 g) were added to a 2 L Erlenmeyer flask that containeddeionized water (1500 mL). The suspension was stirred with a magneticstirrer (2 inch stir bar) and the pH was adjusted to pH 2.1 withconcentrated hydrochloric acid. Pepsin (0.76 9) was added to the flask,which was then stirred in an incubator set to 18° C. Aliquots of thereactions (100 mL) were removed at different times and analyzed forviscosity (Table 2A). The pH of each aliquot was adjusted to between pH3 and pH 3.5 and the samples were stored at 4° C. After the last aliquotwas taken (50 hours), analytical samples (0.7 mL) were combined with pH3.5 acetic acid (1.4 mL) and ultracentrifuged for 1 hour at 45,000 rpmat 4° C. The supernatants and pellets (after being re-suspended in theoriginal volume of buffer) were analyzed for hydroxyproline as shown inTable 2A.

Larger samples of the different fractions (50 mL) were combined with pH3.5 acetic acid (100 mL) and centrifuged at 20,000 rpm for 4 hours at 4°C. The samples were stored at 4° C. for 9-10 days when they were used tomake paper.

                  TABLE 2A                                                        ______________________________________                                        Summary of Results for Example 8A                                                                                ΔTS                                                  Hydroxy-    Hydroxy-                                                                             (% change                                  Sample Viscosity                                                                              Proline in  Proline in                                                                           from Control                               Time   (20 rpm) Supernatant Pellet with no                                    (hrs)  (cps)    (mg/mL)     (mg/mL)                                                                              Addition)                                  ______________________________________                                         3      35      0.10        0.28   15                                          7     400      0.15        0.18   17                                         11     1055     0.23        0.12   21                                         15     1030     0.28        0.06   31                                         26     800      0.29        0.05   35                                         30     745      0.26        0.05   --                                         50     605      0.27        0.04   27                                         ______________________________________                                    

This data demonstrates that collagen was increasingly solubilized inthis reaction up to approximately 15 hours. This was evidenced by theincrease in hydroxyproline in the supernatant, the decrease in thepellet size and hydroxyproline content on centrifugation, and by theinitial increase in viscosity. The increase in soluble collagen wascorrelated with an increase in the tensile strength of the paper towhich the collagen was added, where ΔTS represents the % increase intensile strength above the control paper with no added collagen.

EXAMPLE 9A

Teepak limed splits were centrifuged at 4° C. for 20 minutes at 10,000rpm. The supernatant liquid was removed and the centrifuged limed splits(35 g) were added to a 4 L Erlenmeyer flask that contained deionizedwater (3500 mL). The suspension was stirred with a magnetic stirrer (2inch stir bar) and the pH was adjusted to pH 2.1 with concentratedhydrochloric acid. Pepsin (1.75 g) was added to the flask, which wasthen stirred in an incubator set to 20.5° C. Aliquots of the reactions(200 mL) were removed at different times and analyzed for viscosity(Table 2A). The pH of each aliquot was adjusted to between pH 3 and pH3.5 and the samples were stored at 4° C. until they were used to makepaper.

After 27 hours at 20.5° C., one third of the incubated collagen samplewas removed and stirred at room temperature. The temperature of theincubator was then adjusted to 30° C. and the remainder of the samplewas stirred at this temperature. At specified times, 200 ml samples wereremoved, the pH adjusted, and the samples store at 4° C. as describedabove. After the last aliquot was taken, analytical samples (0.7 mL)were combined with pH 3.5 acetic acid (1.4 mL) and ultracentrifuged for1 hour at 45,000 rpm at 4° C. The supernatants and pellets (after beingre-suspended in the original volume of buffer) were analyzed forhydroxyproline content. The supernatants were also analyzed by sizeexclusion HPLC as shown in Table 3A.

                                      TABLE 3A                                    __________________________________________________________________________    Summary of Results for Example 9A                                             Peak Area                  Hydroxyproline                                     Incubation                                                                         Peak 1                                                                             Peak 2                                                                             Peak 3                                                                             Viscosity                                                                            in Supernatant                                                                       ΔTS.sup.e (%)                         Time (hrs)                                                                         -31 min.                                                                           -34 min.                                                                           ˜45 min.                                                                     (cps at 20 rpm)                                                                      (mg/mL)                                                                              ONP                                                                              OCC                                      __________________________________________________________________________      5.5                                                                              9.0  14.9 5.7  25     0.09   14 27                                       21   17.5 21.0 9.5  375    0.17   27 42                                       23   16.4 24.7 10.1 425    0.17   -- --.sup.f                                 27   9.9  24.7 9.8  650    0.21   28 46                                        30.sup.a                                                                          12.6 20.9 8.7  840    0.23   32 42                                        .sup. 45.5.sup.b                                                                  15.9 23.4 0.7  1095   0.28   37 --                                        30.sup.c                                                                          18.5 30.2 40.2 750    0.20   26 43                                        .sup. 45.5.sup.d                                                                  18.4 24.5 54.4 45     0.30   36 46                                       __________________________________________________________________________     .sup.a This sample was incubated for 27 hours at 20.5° C. and for      hours at rt.                                                                  .sup.b This sample was incubated for 27 hours at 20.5° C. and for      18.5 hours at rt.                                                             .sup.c This sample was incubated for 27 hours at 20.5° C. and for      hours at 30° C.                                                        .sup.d This sample was incubated for 27 hours at 20.5° C. and for      18.5 hours at 30° C.                                                   .sup.e ΔTS = % increase in Tensile Strength of paper over control       (no collagen) made with 1% soluble collagen added to pulps made from Old      News Print (ONP) or Old Corrugated Containers (OCC).                          .sup.f (--) indicates analysis not performed.                            

This data illustrates an increase in soluble collagen throughout thereaction as shown by increases in viscosity and hydroxyprolineconcentration in the supernatant fraction. The increase in solublecollagen is correlated with an increase in the tensile strength of paperto which the collagen was added. Samples kept at 30° C. after 27 hoursof reaction demonstrated progressive conversion of high molecular weightcollagen to degradation products (increase in HPLC peak 3), but in thiscase the lower molecular weight did not result in a similar decrease intensile strength of papers to which it was added. This latter effectindicated that the collagen has a positive effect on the paper even whensome of the material has been digested to relatively low molecularweights. Gel electrophoresis indicates the presence of significantconcentrations of approximately 200,000 dalton collagen andapproximately 100,000 dalton collagen even after reaction at 30° C. for18.5 hours. Thus, in the absence of detergent there may be significantamounts of 300,000 or higher molecular weight material. Substantial highmolecular weight collagen was present as evidenced by the high areas ofHPLC peaks 1 and 2 in samples indicated by footnotes c and d.

EXAMPLE 10A

Two preparations of solubilized collagen were combined as follows. Eachpreparation was made from Teepak limed splits that were centrifuged at4° C. for 20 minutes at 10,000 rpm. The supernatant liquid was removedand the centrifuged limed splits (35 g) were added to a four literErlenmeyer flask that contained deionized water (3500 mL). Thesuspension was stirred with a magnetic stirrer (2 inch stir bar) and thepH was adjusted to pH 2.1 with concentrated hydrochloric acid. Pepsin(1.75 g) was added to the flask, which was then stirred in an incubatorset to 19° C. One preparation was incubated for 31.5 hours (finalviscosity at 20 rpm was 1160 cps) and the other preparation wasincubated for 21 hours (final viscosity at 20 rpm was 1025 cps). The twopreparations were stored at 4° C., with no pH adjustment, for 6 days,then one and a half liters of each preparation were combined in a 4liter flask, stirred to mix, and then rapidly heated to about 30° C. ina water bath. The flask was then stirred in a 32° C. incubator and, atspecified times, 200 ml samples were removed, the pH adjusted to between3.0 and 3.5, and the samples stored at 4° C. The results from thisreaction and the results of tensile tests run on papers made with thesematerials are shown in Table 4A below.

This data demonstrates that, although not all of the collagen wasinitially soluble (hydroxyproline measurements increased throughout thereaction), there was a rapid decrease in collagen number averagemolecular weight throughout the course of the 30° C. reaction period asindicated, for example, by the viscosity decrease and increase in HPLCpeak 3 area. This decrease in molecular weight did not effect the gainin tensile strength until all of HPLC peak 1 (number average molecularweight>300,000 daltons) and nearly all of HPLC peak 2 (number averagemolecular weight ˜300,000 daltons) were converted to smaller fragments.Gel electrophoresis indicated the presence of a small amount of ˜100,000dalton molecular weight collagen even after 25.5 hours at 32° C. Most ofthe collagen has been converted to fragments with number averagemolecular weights less than 100,000 daltons by this time. HPLC analysisof this sample, which is done in the absence of detergent, indicates nopeak 1 and a small of peak 2. The remaining 100,000 dalton numberaverage molecular weight fragments seen on the gel presumably aggregatein the absence of detergent to form the 300,000 dalton triple helix seenas HPLC peak 2. It is this triple helical collagen that appears toimpart the enhanced properties to the paper.

                                      TABLE 4A                                    __________________________________________________________________________    Summary of Results for Example 10A                                            Peak Area                Hydroxyproline                                                                       ΔTS (%                                  Incubation                                                                         Peak 1                                                                             Peak 2                                                                             Peak 3                                                                             Viscosity                                                                          in Supernatant                                                                       Change                                        Time (hrs)                                                                         ˜31 min.                                                                     ˜34 min.                                                                     ˜45 min.                                                                     (cps)                                                                              (mg/mL)                                                                              From Control)                                 __________________________________________________________________________    0    2.1  29.1 --   1260 0.28   +48                                           2    25.8 25.6 12.5 705  0.27   --                                            3    26.8 24.9 19.3 520  0.29   --                                            4    20.4 31.1 37.2 215  0.32   +46                                           5    18.8 28.8 46.1 165  0.29   --                                            6    19.9 31.7 65.3 75   0.34   +37                                           7    13.3 28.8 72.2 35   0.35   +41                                           8    14.7 23.8 83.9 20   0.37   +41                                           9    10.6 22.1 93.9 15   0.37   +47                                           12.5 6.5  16.7 105.5                                                                              10   0.39   +41                                           25.5 0    5.0  127.6                                                                              5    0.38   +30                                           __________________________________________________________________________

EXAMPLE 12A

Reactions of microbial proteases with the collagen from limed splits asdescribed above were as summarized in Tables 5A and 6A:

Microbial proteases were reacted with ground limed splits from twosources at 17° C. A summary of the optimum results with regards toprotease concentration and pH is shown in Table 5A.

                  TABLE 5A                                                        ______________________________________                                        Reaction of Microbial Proteases                                               with Ground Limed Splits                                                                              Maximum  Hrs. to                                                              Viscosity                                                                              Maximum                                      Enzyme       pH         (20 rpm) Viscosity                                    ______________________________________                                        Newlase II (0.08 g)                                                                        2.6        1840     18                                           Quest AP (0.08 g)                                                                          2.6        1535     22                                           AFP 2000 (0.08 g)                                                                          2.6        1415     22                                           EDC-APA (0.08 g)                                                                           2.5        1085     18                                           ______________________________________                                    

                  TABLE 6A                                                        ______________________________________                                        Reaction of Microbial Proteases                                               with Teepak Limed Splits                                                                              Maximum  Hrs. to                                                              Viscosity                                                                              Maximum                                      Enzyme       pH         (20 rpm) Viscosity                                    ______________________________________                                        Newlase II (0.075 g)                                                                       2.6        1386     19                                           Quest AP (0.08 g)                                                                          2.6        945      24                                           EDC-APA (0.08 g)                                                                           2.5        745      20                                           Newlase A (0.04 g)                                                                         2.6        665      23                                           AFP 2000 (0.08 g)                                                                          2.6        515      41                                           EDC-APB (0.08 g)                                                                           3.0        435      39                                           ______________________________________                                    

All of the microbial proteases produce significantly viscous collagensolutions, demonstrating their use for solubilizing collagen from groundlimed splits.

Collagen solutions prepared by the above examples appear to be stable atroom temperature for 12-24 hours, and stability can be enhanced byincreasing solution pH to 3.0-3.5 and/or by reducing the solutiontemperature to 5°-10° C.

The process has demonstrated the feasibility of production of a low-costsoluble collagen product by the substantially complete solubilization ofbeef hide collagen (ground limed-splits). The process can be conductedat near-ambient conditions and is relatively easy to control. Ofparticular interest is the recycle method that reduces the cost of therelatively expensive proteolytic enzymes.

B. SECOND GENERAL EMBODIMENT

The second general embodiment typically utilizes the solubilizedcollagen produced in the first general embodiment or if desiredsolubilized collagen can be obtained from other methods. One majoradvantage of using the solubilized collagen of the first generalembodiment is of course the low cost of the material so produced. Thiscost factor is a major advantage in the paper making art.

The invention improves the strength of recycled paper, conventionalpaper, and mixtures thereof. The invention is especially useful inproducing recycled paper because recycled paper made from recycledcellulosic fibers is generally weaker than paper made from virgincellulose fibers. As used herein the feedstocks in the inventiontypically are: virgin paper pulp which is paper pulp made fromnonrecycled materials; broke which is scrap at the papermaking plant;reclaimed newsprint which is recycled newspaper and similar paper;reclaimed corrugated container which is recycled old corrugatedcontainer and similar material; similar cellulose based papers; andmixtures thereof.

The invention discloses the use of collagen solubilized with enzymes toimprove the strength and other properties of paper type products madefrom cellulose fibers. Typically the method for making a collagenstrengthened paper comprises mixing feedstock with water, or water andcaustic (e.g. NaOH), and mechanically pulping until a pulp slurry isformed. Preferably the pulp slurry has a consistency of about 3 to about6 wt % based on dry pulp solids. The pulp slurry is then diluted to aconsistency of about 1 to about 3 wt % based on dry pulp solids andadjusted to a pH of about 3.5 to about 7.0. Between about 0.1 to about 2dry wt % solubilized collagen is added to the diluted pulp slurry, andthe resulting slurry is mixed at a shear rate and a time effective forinteraction of the diluted pulp slurry solids and soluble collagen,whereby a substantial portion of the solubilized collagen is bound tothe paper pulp to form a collagen-pulp slurry. The collagen-pulp slurryis then diluted, preferably to between about 0.1 and 1 dry wt %consistency, and finally the collagen-pulp slurry is formed into a sheetand dried.

EXAMPLE 1B

Collagen solutions as a coating.

The old newsprint (ONP) or the old corrugated container (OCC) wasshredded and soaked in a 1 percent sodium hydroxide solution overnight.

The shredded material was pulped in a Tappi disintegrator for 15minutes. The pulp was mixed with additional water and a sheet was formedin a Noble and Wood headbox with a Duotex 162-DD-226 forming fabric. Thesheet was wet-pressed on the Noble and Wood and then calendered toincrease density (blotter paper was used on each side and the gap on thecalender rolls was set at 0.76 mils). The sheet was dried on a hot platesurface temperature of about 100° C. under tension for 1 minute.Collagen hydrolysate (MW<2000 daltons) supplied by Secol (Exton, Pa.) orsoluble native collagen (MW>300,000 daltons) supplied by Gattefosse'Corp. (Elmsford, N.Y.) were applied to the sheets of recycled paperusing either a No. 10 or No. 20 wire-wound rod. The coated sheets weredried either in a forced air oven at 100° F. for 10 minutes or allowedto dry at ambient conditions overnight. The coated sheets were evaluatedfor basis weight, burst strength, and tensile properties as reported inTable 1B. This table also details amount of pulp and coating weightused.

Gains of tensile strength were observed in all samples tested, rangingfrom about 125-300 percent over the appropriate control withoutcollagen. While ONP and OCC controls were only approximately 25% asstrong as the Kraft paper standard, several coated samples were asstrong or stronger than the Kraft standard.

                                      TABLE 1B                                    __________________________________________________________________________    Collagen Applied as a Coating                                                                         Physical Characteristics                                                                   Tensile Properties                                                    Mullen         %                                 Sheet Composition       Basis                                                                              Burst          Change                            Sample                                                                            Gms.  Collagen Drying                                                                             Weight                                                                             Strength                                                                          Caliper TS from                              No. Fiber                                                                            Fiber                                                                            Solution/%                                                                          Rod                                                                              Technique                                                                          kg/279 m.sup.2                                                                     MPa mm  TS/BW                                                                             MPa                                                                              Control                           __________________________________________________________________________    CC-1                                                                              5.5                                                                              Kraft                                                                            None  -- --   13.1 .159                                                                              .11 1.30                                                                              17.01                                                                            --                                CC-2                                                                              5.5                                                                              ONP                                                                              None  -- --   12.5 .034                                                                              .18 0.33                                                                              4.08                                                                             --                                CC-4                                                                              5.5                                                                              ONP                                                                              Hydrolysate                                                                         20 oven 17.6 .108                                                                              .13 1.27                                                                              22.34                                                                            +289                              CC-5                                                                              5.5                                                                              ONP                                                                              "     20 air  17.1 .109                                                                              .13 1.06                                                                              18.20                                                                            +151                              CC-6                                                                              5.5                                                                              ONP                                                                              "     10 air  15.3 .092                                                                              .12 1.32                                                                              20.24                                                                            +304                              CC-7                                                                              5.5                                                                              ONP                                                                              "     10 oven 16.8 .108                                                                              .13 1.15                                                                              19.38                                                                            +253                              CC-8                                                                              5.5                                                                              ONP                                                                              Native                                                                              10 air  13.3 .102                                                                              .1I 1.11                                                                              14.71                                                                            +238                              CC-9                                                                              5.5                                                                              ONP                                                                              "     10 oven 14.0 .109                                                                              .12 1.02                                                                              14.22                                                                            +211                              CC-10                                                                             5.5                                                                              ONP                                                                              "     20 air  14.4 .112                                                                              .12 1.09                                                                              15.76                                                                            +235                              CC-11                                                                             5.5                                                                              ONP                                                                              "     20 oven 13.8 .098                                                                              .12 1.10                                                                              15.22                                                                            +238                              CC-12                                                                             4.5                                                                              OCC                                                                              None  -- --   14.4 .055                                                                              .19 0.38                                                                              5.50                                                                             --                                CC-14                                                                             4.5                                                                              OCC                                                                              Hydrolysate                                                                         20 oven 18.2 .178                                                                              .14 1.47                                                                              26.68                                                                            +283                              CC-15                                                                             4.5                                                                              OCC                                                                              "     20 air  18.8 .137                                                                              15  1.09                                                                              20.45                                                                            +185                              CC-16                                                                             4.5                                                                              OCC                                                                              "     10 air  17.7 .161                                                                              .14 1.47                                                                              25.98                                                                            +284                              CC-17                                                                             4.5                                                                              OCC                                                                              "     10 oven 17.2 .161                                                                              .13 1.54                                                                              26.41                                                                            +302                              CC-18                                                                             4.5                                                                              OCC                                                                              Native                                                                              10 air  13.8 .124                                                                              .12 1.02                                                                              14.01                                                                            +166                              CC-19                                                                             4.5                                                                              OCC                                                                              "     10 oven 13.8 .13 .12 1.12                                                                              15.5                                                                             +194                              CC-20                                                                             4.5                                                                              OCC                                                                              "     20 air  14.0 .12 .13 0.86                                                                              12.1                                                                             +126                              CC-21                                                                             4.5                                                                              OCC                                                                              "     20 oven 14.0 .13 .13 0.93                                                                              13.0                                                                             +146                              __________________________________________________________________________

EXAMPLE 2B

Native collagen Added to Pulp in Headbox

The ONP or OCC was shredded and soaked in a 1 percent sodium hydroxidesolution overnight. The material was pulped in a Tappi disintegrator for15 minutes. The pulp was put in the headbox of the Nobel and Wood, andwater at various temperatures (14°-17° C. or 36°-38° C.) was added. ThepH of the slurry was 7. Various amounts of native collagen solution(0.3% solids) were added. The slurry was allowed to settle and stand for4 to 10 minutes. The sheet was formed on a Duotex 162-DD-226 formingfabric. The sheet was wet-pressed on the Noble and wood and thencalendered to increase density. (Blotter paper was used on each side andthe gap on calender rolls was set at 0.762 mm). The sheet was dried on ahot plate for 1 minute. The formed sheets were evaluated for basisweight, burst strength, and tensile properties as reported in Table 2B.This table also details the amount of pulp and collagen additive used.Gains of tensile strength were observed in all samples tested, rangingfrom about 140-350% over the appropriate control without solubilizedcollagen. While ONP and OCC controls were approximately 25% as strong asthe Kraft paper standard, several samples were stronger than the Kraftstandard. No correlation was observed between the amount of collagenadded and the tensile strength improvement.

                                      TABLE 2B                                    __________________________________________________________________________    Collagen Added to Pulp                                                        Sheet Composition       Physical Characteristics                                                                   Tensile Properties                                 Native             Mullen         %                                           Collagen  Slurry                                                                            Basis                                                                              Burst          Change                            Sample                                                                            Gms.  Added                                                                              Headbox                                                                            Temp.                                                                             Weight                                                                             Strength                                                                          Caliper TS from                              No..sup.a                                                                         Fiber                                                                            Fiber                                                                            %    Time °C.                                                                        kg/279 m.sup.2                                                                     MPa mm  TS/BW                                                                             MPa                                                                              Control                           __________________________________________________________________________    CP-1                                                                              5.5                                                                              Kraft                                                                            --   --   --  13.1 .159                                                                              .11 1.30                                                                              16.97                                                                            --                                CP-2                                                                              5.5                                                                              ONP                                                                              --   --   --  12.5 .034                                                                              .18 0.33                                                                              4.07                                                                             --                                CP-5                                                                              5.5                                                                              ONP                                                                              1    4    17.1                                                                              14.3 .092                                                                              .13 1.11                                                                              15.82                                                                            +240                              CP-6                                                                              5.5                                                                              ONP                                                                              1    4    35.9                                                                              13.1 .071                                                                              .12 1.01                                                                              13.19                                                                            +210                              CP-7                                                                              5.5                                                                              ONP                                                                              2    4    15.4                                                                              13.3 .103                                                                              .11 1.48                                                                              19.70                                                                            +353                              CP-8                                                                              5.5                                                                              ONP                                                                              2    4    15.4                                                                              13.9 .081                                                                              .12 0.98                                                                              13.62                                                                            +201                              CP-9                                                                              5.5                                                                              ONP                                                                              1    10   37.6                                                                              13.2 .089                                                                              .12 1.08                                                                              14.20                                                                            +231                              CP-10                                                                             5.5                                                                              ONP                                                                              10   10   35.7                                                                              16.2 .110                                                                              .14 1.07                                                                              17.33                                                                            +227                              CP-11                                                                             4.5                                                                              OCC                                                                              --   --   --  14.4 .055                                                                              .19 0.38                                                                              5.48                                                                             --                                CP-14                                                                             4.5                                                                              OCC                                                                              1    4    14.5                                                                              13.0 .132                                                                              .11 1.48                                                                              19.24                                                                            +289                              CP-15                                                                             4.5                                                                              OCC                                                                              1    4    38.3                                                                              13.5 .111                                                                              .12 0.90                                                                              12.18                                                                            +138                              CP-16                                                                             4.5                                                                              OCC                                                                              2    4    14.9                                                                              13.2 .146                                                                              .11 1.79                                                                              23.68                                                                            +371                              CP-17                                                                             4.5                                                                              OCC                                                                              2    4    37.1                                                                              12.4 .089                                                                              .11 1.06                                                                              13.13                                                                            +179                              CP-18                                                                             4.5                                                                              OCC                                                                              1    10   35.3                                                                              12.7 .096                                                                              .11 1.10                                                                              13.91                                                                            +189                              __________________________________________________________________________     .sup.a All samples except CP1 were soaked in NaOH.                       

EXAMPLE 3B

The examples below illustrate: (1) fiber stocks prepared from oldcorrugated containers (OCC) and old newsprint (ONP); (2) the addition of1% solubilized collagen to those stocks either before or after the papersheet is formed. The feedstocks were used to prepare a lightweight, 13.6kg/279 m², basis weight paper. Some stocks were treated with causticsoda at ambient water temperature. Solubilized collagen was added to thestock chest before paper production in the ratio of 1% of the dry pulpsolids, and mixed for at least 15 min. at a temperature of less than 39°C. The papers were produced as follows:

A. Materials

1. Solubilized collagen prepared as in Example 5A.

2. Post consumer old newsprint (ONP).

3. Liner board (rolls) used as old corrugated container (OCC) did notcontain corrugated medium--Stone Container, Savannah, Ga. The pulpedmaterial is, however, the as if corrugated materials had been used.

4. Concentrated HCL (31%).

B. Equipment:

1. Black Clawson 2.4 m HCVY Hydrapulper 61 cm bottom Vokes rotor anddrive assembly--7570 liter capacity.

2. Sprout-Waldron 30 cm Twin-Flow refiner--1770 rpm equipped with platesD5B053 motor end and D5B054 control end.

3. Sandy Hill Corporation manufactured (1967) Fourdrinier type papermachine with a 97 cm wire width. The table has a forming length of 44.3meters. The slice width is 84 cm and the machine was operated with edgecurls. The machine's press section consisted of two presses, the firstone being a straight through double felted and the second being abottom-felted reversed press. Each press nip is limited to 2.06 MPa. Thebottom press rolls have rubber venta nip covers. The top roll in thesecond press has a stonite cover. The machine's dryer section consistsof two banks of 91 cm diameter dryer cans, seven cans in the firstsection and five cans in the second section. Between the dryer sectionsis a size press arrangement which can be operated as a horizontal or avertical unit. With proper rolls installed, the unit can also be used asa breaker stack. Following the second dryer section is an eight roll,seven nip calender stack. Rolls up to 102 cm in diameter can be wound onthe reel.

C. Paper stock:

100% OCC/530 kg (oven dried)

Old corrugated container was dispersed in ambient temperature waterusing pulper No. 1. The dispersed old corrugated container stock waspumped to a 26,500 liter refiner chest and refined from 644 Canadianstandard freeness (CSF) to 325 CSF in 145 minutes.

100% ONP/552 kg (oven dried)

Old newsprint was dispersed in 66° C. water using pulper No. 1. Thedispersed old newsprint stock was pumped to a 26,500 liter refiner chestand refined from 135 CSF to 107 CSF in 30 minutes.

100% OCC/854 kg (oven dried)

1. Dispersed old corrugated container in ambient temperature water usingpulper No. 1.

2. Pumped dispersed stock to 26,500 liter refiner chest.

3. Refined stock from 638 CSF to 353 CSF in 200 minutes.

100% ONP/871 kg (oven dried)

1. Dispersed ONP in 66° C. temperature water using pulper No. 1.

2. Pumped dispersed stock to 7000 gallon refiner chest.

3. Refined stock from 119 CSF to 99 CSF in 42 minutes.

D. Paper Machine Operations:

Stock from the paper machine chest was pumped via a Fischer-Porter flowcontroller to the suction side of a fan pump. The thick stock was thendiluted with white water to operate the stock flow system. Productionrate on the machine was controlled by the amount of the thick stockflowing into the fan pump. The stock was then pumped through anexplosion chamber manifold into the primary headbox. The headbox wasoperated under vacuum with a top holey roll. Machine speed wasapproximately 175 ft/min. resulting in a paper throughput of about 300lbs./hr.

Wire Set-Up

The forming fabric on the 91 cm Fourdrinier paper machine was a design463Monoflex JDL 145×120 mesh double layer with: forming Board, three 7.6cm diameter table rolls, five foil boxes with four foils each, four flatboxes with adjustable vacuum.

Paper property (e.g. tensile strength, tear strength, burst strength)improvements obtained from the 1% solubilized collagen additions (Table4B). For the mixed fiber stocks, machine direction tensile strengthimprovements were in the range of 25-30% while improvements in the 100%old corrugated containers and old newsprint stocks were in the range of15-20%.

Biological oxygen demand (BOD) effects from the addition of thesolubilized collagen to the mixed fiber were essentially improved overthe plain fiber papers themselves, indicating increased retention ofpaper solids when solubilized collagen was added.

Surface pH measurements of all the papers produced during the trialswere acidic even though the water at the papermaking facilities averagedpH 7 for the month of January, which is typical for the water supply.The solubilized collagen-containing papers showed somewhat lower pHs(more acidic) than the other papers. For some eventual end-useapplications, it may eventually be desirable to bring the pHs of thesolubilized collagen-containing papers to a more neutral level, afterthe papers have been formed.

                                      TABLE 3B                                    __________________________________________________________________________    Properties of Control Papers from Example No. 3B.sup.(1)                      Sample                                                                            Paper        Tensile St.                                                                         MD  Tear      Mullen                                   No. Identification                                                                        pH                                                                              BW MD CD TS/BW                                                                             MD CD Caliper                                                                           Burst                                                                             BOD                                  __________________________________________________________________________    Control Papers                                                                16A 100% OCC                                                                              --                                                                              14.2                                                                             -- -- 1.68                                                                              71.3                                                                             76.1                                                                             .10 .189                                                                              380                                  19A 75125 OCC/ONP                                                                         --                                                                              -- -- -- 1.49*                                                                             -- -- --  .151                                                                              --                                   17CA                                                                              50/50 OCC/ONP                                                                         --                                                                              14.1                                                                             -- -- 1.23                                                                              53.7                                                                             58.8                                                                             .12 .113                                                                              170                                  18A 25/75 OCC/ONP                                                                         --                                                                              -- -- -- 1.08*                                                                             -- -- --  .105*                                                                             --                                   15A 100% ONP                                                                              --                                                                              14.3                                                                             -- -- .93 37.1                                                                             41.1                                                                             .14 .096                                                                              190                                  __________________________________________________________________________     .sup.(1) Abbreviations and units are as follows: BW = Basis Wt., kg/279       m.sup.2 (lbs./3000 ft.sup.2) of paper; TS = Tensile Strength, MPa; MD =       Machine Direction; CD = Cross Direction; Tear = Tear Strength, grams;         Caliper, mm; Mullen Burst, MPa; BOD = Biological Oxygen Demand, mg/liter.     *Estimates used for comparison of additives in Table 4B.                 

                                      TABLE 4B                                    __________________________________________________________________________    Properties of Experimental Papers from Example No. 3B.sup.(1)                 Sample                                                                            Paper         Tensile                                                                             MD       Tear.sup.(2)                                                                             Mullen                            No. Indentification                                                                       pH BW MD CD TS/BW                                                                             ΔTS/BW                                                                       MD  CD Caliper                                                                           Burst.sup.(2)                                                                     BOD.sup.(2)                   __________________________________________________________________________    Experimental Papers with 1% solubilized collagen                              16  100% OCC                                                                              (3.9)                                                                            13.3                                                                             25.79                                                                            11.9                                                                             1.94                                                                              15%  54.0                                                                              71.4                                                                             .10 .170                                                                              260                                                            (24%)                                                                             (6%)   (10%)                                                                             32%                           19  75/25 OCC/ONP                                                                         (3.9)                                                                            13.3                                                                             25.87                                                                            10.73                                                                            1.91                                                                              29%  58.8                                                                              68.6                                                                             .11 .145                                                                              --                                                                         (4%)                             17C 50/50 OCC/ONP                                                                         (3.9)                                                                            13.9                                                                             22.52                                                                            11.02                                                                            1.62                                                                              32%  56.4                                                                              60.0                                                                             .12 .133                                                                              160                                                            5%  2%     18%  6%                           18  25/75 OCC/ONP                                                                         (3.9)                                                                            14.1                                                                             19.01                                                                            9.14                                                                             1.35                                                                              25%  47.6                                                                              51.2                                                                             .13 .122                                                                              --                                                                        17%                               15  100% ONP                                                                              (3.9)                                                                            14.2                                                                             15.49                                                                            8.08                                                                             1.09                                                                              18%  37.6                                                                              40.8                                                                             .13 .111                                                                              150                                                            1%  (1%)   16% 21%                           __________________________________________________________________________     .sup.(1) Abbreviations and units are as follows: BW = Basis Wt., kg/279       m.sup.2 (lbs./3000 ft.sup.2) of paper; TS = Tensile Strength, MPa; MD =       Machine Direction; CD = Cross Direction; Tear = Tear Strength, grams;         Caliper, mm; Mullen Burst, MPa; BOD = Biological Oxygen Demand, mg/liter.     Basis weight is the paper weight in kg/279 m.sup.2 (lbs. per 3000 sq. ft.     of paper                                                                      .sup.(2) Percentage numbers shown in these columns indicate that the data     from the experimental papers were increased/decrcased from the data of th     equivalent daily control papers.                                         

EXAMPLE 4B

Mixing of Soluble Collagen and Pulp Fiber Prior to Headbox

Seven aliquots of solubilized collagen samples, prepared as described inExample 8A and collected after 16 or 20 hours, were pooled to yield acollagen solution with approximately 3.5 mg collagen solids/ml and aviscosity of 1150 cps at 20 rpm. Aliquots of this solution were dilutedeither 8-, 4-, or 2-fold with distilled water, or used undiluted, togive a range of concentrations of approximately 0.44, 1.75 and 3.5 mgcollagen solids/ml.

Pulp slurries were prepared from ONP and OCC paper stocks at 3%consistency by shredding the materials, soaking them in 1% NaOHovernight, rinsing the soaked solids in tap water, and pulping therinsed solids in a Tappi disintegrator for 15 minutes.

The pulp slurry was heated on a hot plate with manual stirring toapproximately 120°-125° F. An aliquot of the heated pulp slurry (183 g)was combined with an aliquot of one of the diluted collagen solutions(63 g), and the combined collagen-pulp slurry was stirred by ablade-type mixer for 15 min. The resulting consistency of the pulp inthe slurry was about 2.2%. The collagen solids to pulp solids ratio forthese experiments therefore were approximately 0.5%, 1%, 2% and 4%. Theinitial temperature of the pulp-collagen slurry was approximately 106°F. +/- 3° F. (41° C. +/- 2° C.), and this temperature decreased toapproximately 95° F. by the end of the stirring period.

At the end of the mixing period, the collagen-pulp slurry was put in theheadbox of the Nobel and Wood handsheet system and collected by drainagethrough a Duotex 162-DD-226 forming fabric. The formed sheet was wetpressed, then calendered between blotter paper with the calender gap setat 30 mils. The sheet was then dried on a hot plate under tension for 1min. Handsheets were equilibrated overnight in a controlled environmentroom (72° F./50 % RH), then evaluated for basis weight (BW) and tensilestrength (TS). Three sheets were prepared and tested for each samplecondition. Results are summarized in Table 5B.

This example illustrates that increasing concentrations of dissolvedcollagen, when added to constant amounts of secondary pulp fiber,generally results in increases in tensile strength of sheets formed fromthe combination. The only exception in the data of Table 4B was for theOCC sheets with 0.5% added collagen, which yielded a corrected averageTensile Strength (TS/BW) slightly lower than the

                  TABLE 5B                                                        ______________________________________                                        Summary of Handsheet Properties for Example 4B Tests                               Collagen                                                                      Added      Avg. BW*  Avg.   Avg.   ΔTS                             Pulp %          (lb/3000 ft.sup.2)                                                                      TS*    TS/BW* (%)                                   ______________________________________                                        OCC   0**       31.0      1330   42.9   --                                    OCC    0.5      36.3      1430   39.4   -8.1                                  OCC  1          35.2      1790   50.9   +19                                   OCC  2          34.9      1840   52.7   +23                                   OCC  4          35.7      1930   54.1   +26                                   ONP   0**       28.0      1370   48.9   --                                    ONP    0.5      32.7      1740   53.2   +8.8                                  ONP  1          33.2      1930   58.1   +19                                   ONP  2          31.8      2020   63.5   +30                                   ONP  4          34.1      2400   70.4   +44                                   ______________________________________                                         *Average of 3 handsheets                                                      **Control handsheets                                                          BW = basis weight, lbs/3000 sq. ft. of paper;                                 TS = tensile strength                                                    

OCC control tensile Strength (-8.1%). This apparently inconsistent valueis believed to have resulted from the consistently higher Basis Weightsof the papers containing collagen (approximately 15% higher than controlfor the OCC sheets), which is believed to have resulted from theincreased retention of pulp fines (small pulp fibers) in these samples.More fines, which would generally produce weaker papers, would tend tosuppress the strength of the resulting paper, as in the 0.5%/OCC datumcited. This data clearly illustrates the general property of thecollagen additive as a retention aid in paper formation.

As more collagen was added to either pulp slurry, the resulting paperstrength increased, but the gain of strength was not linearlyproportional to the amount of collagen added; the strength enhancementtended to decrease with increasing collagen/pulp ratio. This observationis consistent with a process of interaction of soluble collagenmolecules with pulp fibers that results in saturation of the fibersurfaces with adsorbed collagen molecules. The strength enhancementobserved in this binding process is believed to result from inter-fiberbridges formed by soluble collagen molecules; saturation of the fibersurfaces with bound collagen would tend to limit the extent of suchinter-fiber bridges, and thereby limit the maximum strength enhancementimparted by this process.

In the example cited, the apparent saturation process observed isinterpreted as confirmation that interactions between soluble collagenmolecules and pulp fiber surfaces is the predominate mechanism ofstrength enhancement, as opposed to the directly additive strengthenhancement behavior that would be anticipated if these were nointeractions between two populations of insoluble fibers mixed in thesame proportions. In the examples summarized in Table 5B, the OCC fibersappeared to saturate at a lower collagen-to-pulp-solids ratio than didthe ONP fibers.

This example also illustrates that the strength enhancement due tointeractions between soluble collagen and pulp fibers can occur attemperatures above 40° C., above which collagen molecules wouldgenerally be expected to denature thermally. Previous citations haveindicated that collagen addition to paper must be made below thisdenaturation temperature (G. Sauret et al, Le collagne ans lafabrication du papier, Revue A.T.P.I., Vol 33, No. 8, Octobre 1979, pp374-365). In a preliminary series of experiments (data not includedherein), it was observed that if the pulp slurry and collagen solutionwere mixed at about 40° C. or above at low pulp slurry consistencies(e.g., 0.5% pulp solids), then the collagen tended to precipitate fromsolution before binding to the pulp fibers, leading to unsatisfactory(speckled) paper surfaces and no significant enhancement of tensilestrength. On the other hand, if the pulp slurry and collagen solutionswere mixed at higher pulp consistencies (e.g., 2.2% pulp solids as inTable 5B), the collagen does not precipitate and is successfully boundto the pulp fibers.

An additional example on the effect of temperatures in excess of 30° oncollagen preparation is provided by the following example. USDA groundlimed splits (0.06 inch cutting head) were centrifuged at 4° C. for 20minutes at 10,000×g and the supernatant liquid was removed. Thecentrifuged limed splits were added in 7.5 g portions to two 1 LErlenmeyer flasks that each contained deionized water 750 mL). Thesuspensions were stirred with a magnetic stirrer (2 inch stir bar), thepH was adjusted to pH 2.1 using concentrated hydrochloric acid, and 0.19g pepsin was added to each flask. One flask was stirred at 19° C. andthe other flask was stirred at 32° C. After 60 hours, the pH of theflasks were adjusted to Proximately 3.5 and the viscosities weremeasured at 20 rpm. The viscosity of the 19° C. reaction was 620 cps andthe viscosity of the 32° C. reaction was 10 cps.

Both collagen preparations were added to pulp (collagen is approximately1% of pulp) and the ability of these preparations to improve theproperties of paper were measured. The preparation made at 19° providedno tensile strength/basis weight enhancement.

This shows that completely hydrolyzed soluble collagen does not appearto contribute to enhancement of tensile strength. The measurement ofviscosity below about 20 cps does not appear sufficient to predict thedegree of strength enhancement of paper mode with these solutions.

While the various examples above have focused on papermaking theinvention could also be used in the making of various products such asmolded products or paperboard where a cellulosic pulp can be bonded bysolubilized collagen.

Various types of water such as Columbus, Ohio tap water; Savannah, Ga.tap water; whitewater from the papermaking process; and whitewaterreduced in solids content were used; thus, it appears that the type ofwater is not critical in the invention for either the collagen makingprocess or the papermaking process and a wide latitude for watersupplies is possible.

While the forms of the invention herein disclosed constitute presentlypreferred embodiments, many others are possible. It is not intendedherein to mention all of the possible equivalent forms or ramificationsof the invention. It is to be understood that the terms used herein aremerely descriptive, rather than limiting, and that various changes maybe made without departing from the spirit of the scope of the invention.

We claim:
 1. A method for making a collagen strengthened cellulosicsheet comprising:a. mixing a cellulosic material selected from the groupconsisting of virgin paper pulp, broke, reclaimed newsprint, reclaimedcarton container, or a mixture thereof with a solution comprising water,or water and NaOH, and mechanically pulping until a pulp slurry isformed having a consistency of about 3 wt % to about 6 wt % based on drypulp solids; b. diluting said pulp slurry to a consistency of about 1 wt% to about 3 wt % based on dry pulp solids and adjusting pH to about 3.5to about 7.0; c. adding an alum/rosin additive to said pulp slurry afterStep a or to said diluted pulp slurry after step b; d. forming saiddiluted pulp slurry containing alum rosin into a sheet; e. coating oneor both sides of said sheet with soluble collagen having a numberaverage molecular weight of at least 300,000 daltons; and f. drying saidsheet.
 2. A method for making a collagen strengthened cellulosic productcomprising the steps of:a. providing a cellulosic pulp slurry; b.forming the cellulosic pulp slurry into a product of desired shape; c.applying soluble collagen having a number average molecular weight of atleast 300,000 daltons to said cellulosic product; and, d. drying saidcellulosic product.
 3. A method as set forth in claim 2, furthercomprising adding an internal sizing additive to said cellulosic pulpslurry to permit sizing to occur.
 4. A method as set forth in claim 3,wherein said internal sizing additive is an alum/rosin additive.
 5. Amethod as set forth in claim 2, wherein said cellulosic product is acellulosic sheet.
 6. A method as set forth in claim 5, whereby saidapplying step is accomplished by coating said cellulosic sheet with thesoluble collagen.
 7. A method as set forth in claim 5, whereby saidapplying step is accomplished by spraying said cellulosic sheet with thesoluble collagen.
 8. A method as set forth in claim 2, wherein saidsoluble collagen has a number average molecular weight above 1,000,000daltons.
 9. A method as set forth in claim 2, wherein said cellulosicpulp slurry comprises cellulosic material selected from the groupconsisting of virgin paper pulp, broke, reclaimed newsprint, reclaimedcarton container, or a mixture thereof.
 10. A method as set forth inclaim 2, wherein said cellulosic pulp slurry is prepared by a methodcomprising:a. mixing cellulosic material selected from the groupconsisting of virgin paper pulp, broke, reclaimed newsprint, reclaimedcarton container, or a mixture thereof, with a solution comprisingwater, or water and caustic; and b. pulping said mixture until acellulosic pulp slurry is formed with appropriate consistency.
 11. Amethod as set forth in claim 10, whereby said cellulosic pulp slurry hasa consistency of about 3 wt % to about 6 wt %, based on dry pulp solids.12. A method as set forth in claim 10, further comprising adding aninternal sizing additive to the cellulosic pulp slurry after pulping.13. A method as set forth in claim 12, wherein said internal sizingadditive is an alum/rosin additive.
 14. A method as set forth in claim10, further comprising diluting said cellulosic pulp slurry to aconsistency of about 1 wt % to about 3 wt % based on dry pulp solids andadjusting pH to about 3.5 to about 7.0.
 15. A method as set forth inclaim 14, further comprising adding an internal sizing additive to saiddiluted cellulosic pulp slurry instead of after pulping.
 16. A method asset forth in claim 15, wherein said internal sizing additive is analum/rosin additive.
 17. A method as set forth in claim 10, wherein saidcaustic is NaOH.
 18. A method as set forth in claim 17, wherein saidNaOH solution has a concentration of about 0.25 wt % to above 1.00 wt %based on dry weight of solids and a pH range of 10-14.
 19. A method asset forth in claim 14, whereby said pH is adjusted with an acid selectedfrom the group consisting of muriatic acid, HCl, HNO₃, H₂ SO₄, andacetic acid.